Spectrofluorometric studies on the binding interaction of bioactive imidazole with bovine serum albumin: A DFT based ESIPT process

Article abstract of DOI:10.1016/j.saa.2011.04.049

Bioactive imidazole derivatives were synthesized and characterized by NMR spectra, mass and CHN analysis. An excited state intramolecular proton transfer (ESIPT) process in hydroxy imidazole has been studied using emission spectroscopy. In hydrocarbon sol

Full text of DOI:10.1016/j.saa.2011.04.049

Spectrochimica Acta Part A 79 (2011) 1240–1246
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectrofluorometric studies on the binding interaction of bioactive imidazole
with bovine serum albumin: A DFT based ESIPT process
Jayaraman Jayabharathi∗, Venugopal Thanikachalam, Kanagarathinam Saravanan,
Marimuthu Venkatesh Perumal
Department of Chemistry, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioactive imidazole derivatives were synthesized and characterized by NMR spectra, mass and CHN
analysis. An excited state intramolecular proton transfer (ESIPT) process in hydroxy imidazole has been
studied using emission spectroscopy. In hydrocarbon solvent, the tautomer emission predominates over
the normal emission and in alcoholic solvent like ethanol; a dramatic enhancement of normal emission
is observed which was due to increased solvation. DFT calculation on energy, charge distribution of the
rotamers in the ground and excited states of the imidazole derivative were performed and discussed.
PES calculation indicates that the energy barrier for the interconversion of two rotamers is too high in
the excited state than in the ground state. The interaction between bioactive imidazole derivative and
bovine serum albumin (BSA) was investigated.
Received 7 March 2011
Received in revised form 26 March 2011
Accepted 15 April 2011
Keywords:
Bioactive imidazole
ESIPT
PES
FRET
© 2011 Elsevier B.V. All rights reserved.
Binding distance
Binding constant
1. Introduction
tant method to study the interaction of substances with protein
because of its accuracy, sensitivity, rapidity and convenience of
Bio-active imidazole derivatives attracted considerable atten-
tion because of their unique optical properties [1,2] and the
imidazole nucleus forms the main structure of human organ-
isms, i.e., the amino acid histidine, Vitamin B12, a component
of DNA base structure and also have analytical applications uti-
lizing their fluorescence and chemiluminescence properties [3].
Excited state intramolecular proton transfer (ESIPT) phenomena
have been investigated [4–8] in the past decades due to the practical
applications of ESIPT exhibiting molecules as laser dyes [4], photo
stabilizers [5], fluorescent probes in biology [6] and light-emitting
materials for electroluminescent devices [7–9]. ESIPT occurs in
aromatic molecules having a phenolic hydroxy group with an
intramolecular hydrogen bond to the nearby hetero atom of the
same chromophore.
usage.
In the present paper, we will focus the light on the photophys-
ical studies of 2-aryl imidazole derivatives (1–6), ESIPT process of
hydroxy imidazole, density functional theory (DFT) calculation on
energy, dipole moment and PES studies of various rotamers. We
also exploited the detailed investigation of BSA–imidazole associa-
tion (binding parameters and the effect of imidazole on the protein
conformation) using fluorescence and UV–vis absorption studies.
2. Experimental
2.1. Materials and methods
Serum albumins are the most abundant proteins in plasma
[10,11], among the serum albumins, BSA has a wide range of phys-
iological functions involving the binding, transport and delivery of
fatty acids, porphyrins, steroids, etc. Steady state and time-resolved
fluorescence spectroscopy are used for analyzing the binding prop-
erties of BSA and drugs and also to study the structural information
of protein cavities [12–14]. Fluorescence quenching is an impor-
Butane-2,3-dione (Sigma–Aldrich Ltd.), 4-fluoro benzaldehyde
(S.D. fine.), 4-fluoro salicylaldehyde, 4-fluoro-2-methoxy benzalde-
hyde, 3-methyl aniline, 2,5-dimethyl aniline and all other reagents
used without further purification. Bovine serum albumin (BSA) was
obtained from Sigma–Aldrich Company, Bangalore. All BSA solu-
tion was prepared in the Tris–HCl buffer solution (0.05 mol L−1 Tris,
0.15 mol L−1 NaCl, pH 7.4) and was kept in the dark at 303 K. Tris
base (2-amino-2-(hydroxymethyl)-1,3-propanediol) had a purity
of not less than 99.5% and NaCl, HCl and other starting materials
were all of analytical purity and doubly distilled water was used
throughout.
∗
Corresponding author. Tel.: +91 9443940735.
E-mail address: jtchalam2005@yahoo.co.in (J. Jayabharathi).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.04.049

J. Jayabharathi et al. / Spectrochimica Acta Part A 79 (2011) 1240–1246
1241
2.4.3. 4,5-Dimethyl-1-(2,5-dimethylphenyl)-2-phenyl-1H-
imidazole (3)
Yield: 47%. 1H NMR (400 MHz, CDCl3): ı 1.85 (s, 3H), 1.90 (s,
3H), 2.29 (s, 3H), 2.34 (s, 3H), 7.00 (s, 1H), 7.15 (m, 5H), 7.36 (m,
2H). 13C NMR (100 MHz, CDCl3): ı 9.10, 12.79, 16.84, 20.79, 124.94,
126.99, 127.45, 128.01, 129.81, 131.08, 132.76, 133.54, 136.90,
144.49, 160.94, 163.01. Anal. calcd. for C19H20N2: C, 82.57; H, 7.29;
N, 10.14. Found: C, 82.01; H, 6.98; N, 9.86. MS: m/z 276.30, calcd.
276.16.
2.4.4. 2-(4,5-Dimethyl-1H-imidazol-2-yl) 5-fluorophenol (4)
Yield: 44%. Anal. calcd. for C11H11N2OF: C, 64.07; H, 5.38; N,
13.58. Found: C, 64.82; H, 5.92; N, 14.04. 1H NMR (400 MHz, CDCl3):
ı 2.27 (s, 3H), 2.30 (s, 3H), 6.80–7.14 (aromatic protons), 10.30
(s, 1H), 12.20 (s, 1H). 13C (100 MHz, CDCl3): ı 9.42, 12.36, 116.40,
121.00, 127.23, 130.53, 146.08, 148.21, 154.30. MS: m/z 206.5, calcd.
206.22.
Scheme 1. Synthesis of imidazole (1–6).
2.2. Optical measurements and composition analysis
2.4.5. 2-(5-Fluoro-2-methoxyphenyl)-4,5-dimethyl-1-phenyl-
1H-imidazole
(5)
Yield: 47%. Anal. calcd. for C18H17N2OF: C, 72.95; H, 5.78; N,
9.45. Found: C, 73.10; H, 5.97; N, 9.89. 1H NMR (400 MHz, CDCl3):
ı 2.01 (s, 3H), 2.23 (s, 3H), 3.80 (s, 3H), 6.78–7.45 (aromatic pro-
tons), 13C (100 MHz, CDCl3): ı 9.43, 12.71, 55.28, 116.20, 118.00,
121.00, 127.23, 130.00, 134.48, 137.90, 144.31, 155.28. MS: m/z
296.03, calcd 296.34.
NMR spectra were recorded on a Bruker 400 MHz. The UV–vis
spectra and photoluminescence (PL) spectra were measured on
UV–vis spectrophotometer (Perkin Elmer, Lambda 35) and fluores-
cence spectrometer (Perkin Elmer LS55), corrected for background
due to solvent absorption. MS spectra were recorded on a Varian
Saturn 2200 GCMS spectrometer. The quantum yield (˚p), radiative
(kr) and non-radiative (knr) deactivation pathways are calculated to
be: ˚unk = ˚std(Iunk/Istd)(Astd/Aunk)(ꢀunk/ꢀstd)2, where Фunk, Фstd
,
Iunk, Istd, Aunk, Фunk and Фstd are the fluorescence quantum yields,
the integration of the emission intensities, the absorbances at the
excitation wavelength and the refractive indexes of the corre-
sponding solution of the 2-aryl imidazole derivatives (1–6) and the
standard, respectively.
2.4.6. 4-(4,5-Dimethyl-1-(2,5-dimethylphenyl)-1H-imidazol-2-
yl)phenol
(6)
Yield: 49%. 1H NMR (400 MHz, CDCl3): ı 1.84 (s, 3H), 1.89 (s,
3H), 2.28 (s, 3H), 2.35 (s, 3H), 4.6 (s, 1H) 6.85 (m, 2H), 6.97 (s,
1H), 7.17 (d, 2H), 7.33 (m, 2H).13C NMR (100 MHz, CDCl3): ı 9.12,
12.83, 16.87, 20.85, 115.19, 124.97, 127.46, 128.92, 130.00 133.99,
136.98, 143.74, 161.01, 163.48. Anal. calcd. for C19H20N2O: C, 77.52;
H, 6.51; N, 9.52. Found: C, 77.01; H, 6.02; N, 9.21. MS: 292.16, calcd.
292.00.
2.3. Computational details
Quantum mechanical calculations were carried out using
Gaussian-03 program [15]. As the first step of our DFT calculation,
the geometry taken from the starting structure was optimized.
2.5. Principles of fluorescence quenching
2.4. General procedure for the synthesis of 2-aryl imidazole
derivatives (1–6)
Fluorescence quenching [25] is described by the Stern–Volmer
equation:
The experimental procedure was used as the same as described
in our recent papers [16–24]. The 2-aryl imidazole derivatives (1–6)
were synthesized from an unusual four components assembling
of butane-2,3-dione, ammonium acetate, substituted anilines and
substituted benzaldehydes (Scheme 1).
F0
= 1 + Kqꢁ0[Q] = 1 + KSV[Q]
(1)
F
where F0 and F are the fluorescence intensities before and after
the addition of the quencher, respectively. Kq, KSV, ꢁ0 and [Q] are
the quenching rate constant of the bimolecular, the Stern–Volmer
dynamic quenching constant, the average lifetime of the bimolec-
ular without quencher (ꢁ0 = 10−8 s) and the concentration of the
quencher, respectively. Obviously, Kq = KSVꢁ0, hence, Eq. (1) was
applied to determine KSV by linear regression of a plot of F0/F versus
[Q].
2.4.1. 4,5-Dimethyl-2-phenyl-1-m-tolyl-1H-imidazole (1)
Yield: 53%. 1H NMR (400 MHz, CDCl3): ı 2.00 (s, 3H), 2.28 (s, 3H),
2.36 (s, 3H), 6.97 (d, 2H), 7.18 (m, 4H), 7.23 (d, 1H), 7.33 (m, 2H).
13C NMR (100 MHz, CDCl3): ı 9.57, 12.75, 21.28, 125.00, 127.94,
128.40, 129.20, 130.93, 133.48, 137.94, 139.55, 145.06. Anal. calcd.
for C18H18N2: C, 82.41; H, 6.92; N, 10.68. Found: C, 82.22; H, 6.74;
N, 10.46. MS: m/z 262.20, calcd. 262.15.
2.6. Calculation of binding parameters
2.4.2. 2-(4-Fluorophenyl)-4,5-dimethyl-1-m-tolyl-1H-
imidazole (2)
Apparent binding constant kA and binding sites n [26] can be
obtained from
Yield: 55%. 1H NMR (400 MHz, CDCl3): ı 2.00 (s, 3H), 2.27 (s, 3H),
2.36 (s, 3H), 6.86 (s, 2H), 6.94 (s, 1H), 6.95 (s, 1H), 7.22 (d, 1H), 7.29
(m, 3H). 13C NMR (100 MHz, CDCl3): ı 9.53, 12.69, 21.26, 114.86,
115.08, 124.94, 125.34, 127.16, 128.35, 129.31, 129.81, 133.44,
137.73, 139.69, 144.19, 160.99, 163.45. Anal. calcd. for C18H17N2F:
C, 76.12; H, 6.11; N, 9.99. Found: C, 76.06; H, 5.96; N, 9.83. MS: m/z
281.30, calcd. 280.14.
log(F0 − F)
= log KA + n log[Q]
(2)
F
where F0 and F are the fluorescence intensities before and after the
addition of the quencher, [Q] is the total quencher concentration. By
the plot of log(F0 − F)/F versus log[Q], the number of binding sites
n and binding constant KA can be obtained.

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J. Jayabharathi et al. / Spectrochimica Acta Part A 79 (2011) 1240–1246
Table 1
Absorption and photoluminescence spectral data of 1–6 in various solvents.
Solvent
Absorptiona (ꢂ, nm) (log ε)
Emissionb (ꢂ, nm)
1
2
3
4
5
6
1
2
3
4
5
6
1,4-Dioxane
Benzene
Chloroform
Ethyl acetate
Dichloromethane
Ethanol
274.0 (3.20)
277.5 (3.83)
283.0 (4.06)
284.0 (4.26)
286.5 (4.33)
288.0 (3.89)
291.0 (4.07)
296.0 (4.17)
276.0 (4.07)
280.0 (3.88)
285.0 (4.48)
288.0 (4.24)
290.0 (4.16)
291.0 (3.93)
293.0 (4.07)
297.0 (4.17)
274.0 (4.11)
279.0 (3.94)
281.0 (4.50)
284.0 (4.28)
288 (4.28)
293.0 (4.17)
297.0 (4.26)
301.5 (3.07)
276 (4.08)
277 (3.98)
280 (4.18)
282 (4.33)
286 (4.37)
290 (3.74)
298 (4.11)
302 (4.13)
278 (4.20)
280 (4.07)
281 (4.31)
282 (4.25)
285 (4.29)
291 (3.88)
297 (4.32)
303 (4.31)
275.0 (4.09)
278.0 (4.03)
287.0 (4.30)
290.0 (4.03)
292.0 (4.22)
295.0 (4.11)
299.0 (4.04)
303.0 (4.09)
360
363
371
378
386
391
398
406
363
370
372
377
381
383
388
399
362
368
371
375
384
388
393
401
365, 400 (sh)
367
372
376
380
388
392
403
360
362
365
366
368
370
382
390
362
366
374
377
384
385
388
397
Methanol
Acetonitrile
a
UV–vis absorption measured in solution concentration = 1 × 10−5 M.
Photoluminescence measured in solution concentration = 1 × 10−4 M.
b
However, in the case of hydroxyl solvent (EtOH), a short
wavelength emission band appears for 4 which is absent in the flu-
orescence spectra of 1–3, 5 and 6 (Fig. 2). This result corresponds
to the data obtained earlier [27,28] for compounds demonstrat-
ing ESIPT and can be explained by the presence of intermolecular
hydrogen bonding with solvent molecule leading to the stabiliza-
tion of solvated isomer IV in which ESIPT is impossible.
For better understanding the ESIPT mechanism in 4, we per-
formed DFT calculation of electron density for the keto and enol
isomers of 4 in the ground and the excited states (Table 2) which
reveal, excitation of enol isomer leads to an increase in electron
density at N(5) atom and decrease at oxygen atom resulting in ESIPT
and formation of the excited keto isomer in excited state, excited
keto isomer emits luminescence and returns to the ground state
keto form, which is characterized by a large positive charge at N(5)
atom and negative charge at oxygen atom. As a result, a reverse
process occurs in the ground state keto isomer producing an initial
molecule in enol form.
Fig. 1. Excitation spectra of 2-aryl imidazole derivatives 1–6 in ethanol.
3. Results and discussion
All 2-aryl imidazole derivatives (1–6) fluoresce strongly in solu-
tions at room temperature. Their luminescence excitation spectra
(Fig. 1) are in coincide with their absorption spectra and differ from
their emission spectra (Fig. 2), therefore, the fluorescence of the 2-
aryl imidazole derivatives (1–6) was taken for discussion (Table 1).
It may be suggested that in aprotic solvents, the hydroxy 2-
aryl imidazole (4) exists as two different intramolecular hydrogen
bonded isomers I and II (Fig. S1). Excitation of the isomer II should
lead to the formation of the keto-isomer III due to ESIPT (Fig. S1),
while excitation of the isomer I must yield the normal emission.
But, we found out that the fluorescence spectra of 4 in dioxane
contain an abnormal Stokes-shifted emission band and one small
shoulder peak at higher wavelength which reveal that only the iso-
mer II is stable under these conditions. Stokes shift is important for
a fluorescent sensor, the higher Stokes shift value supplies very low
background signals and resultantly allows the usage of the material
in construction of a fluorescence sensor [27].
3.1. Driving force for ESIPT process
The existence of intramolecular hydrogen bond in hydroxy imi-
dazole 4 is confirmed by the presence of the singlet at 10.30 ppm
in the 1H NMR spectra which is a typical signal for hydrogen
bonded hydrogen atom. In order to reveal the contribution of the
intramolecular hydrogen bonding in the hydroxy imidazole 4 to
their optical properties, the fluorescence spectra of compound 4
and its methoxy derivative 5 were measured in dioxane solvent
under identical condition (Fig. 3). A dual fluorescence was detected
for 4 with emission peak centered at 365 and 400 nm respectively.
The emission peak at shorter wavelength at 365 nm is assigned
to rotamer I and longer wavelength band at 400 nm is assigned
to rotamer II whereas compound 5 exhibits emission peak only at
360 nm, absence of additional peak at longer wavelength confirms
that absence of intramolecular hydrogen bond in 5 which further
evident that intramolecular hydrogen bond is the essential driv-
ing force for ESIPT and the dual fluorescence behaviour of hydroxy
imidazole 4.
3.2. Potential energy curve
The ground and excited state geometries of the three rotamers,
I, II, and III of 4 were optimized using the DFT/6-31G(d,p) and CIS
Table 2
Electron density of atoms N(5) and O for 4.
Atom
II
II*
III
0.362
−0.786
N(5)
O
−0.398
−0.581
−0.482
−0.503
*
Fig. 2. Emission spectra of 2-aryl imidazole derivatives 1–6 in ethanol.
Excited state isomer II

J. Jayabharathi et al. / Spectrochimica Acta Part A 79 (2011) 1240–1246
1243
Fig. 5. Absorption spectra of BSA in the presence of imidazole (6) (a–e) and absence
of imidazole (6) (f).
Fig. 3. Fluorescence spectra of 4 and 5 in dioxane.
Table 3
Relative energies (eV) and dipole moments (D) of 4 in the ground and excited state
for rotamers I, II and III.
Rotamers
Ground state
Excited state
ꢃ (D)
E (eV)a
E (eV)a
ꢃ (D)
I
II
III
3.82
3.03
5.69
0.06 (0.00)
0.10 (0.06)
0.68 (0.42)
4.26 (3.62)
4.32 (3.96)
4.01 (3.36)
9.02
8.68
5.01
a
Relative energies are calculated with respect to the ground state minimum
energy form in ethanol. Values in the parenthesis are recorded in ethanol.
methods [28–30] respectively (Table 3). In the excited state the
nitrogen atom becomes richer in electrons than the hydroxylic
oxygen atom. This redistribution of the ␲-electron densities in the
excited electronic state is the driving force for the intramolecular
proton transfer from the hydroxylic group to the nitrogen atom.
The potential energy (PE) curves (Fig. 4a and b) reveal that the
energy barrier for the interconversion of isomers I and II of 4 in
the ground state is 4.9 kcal/mol and 3.2 kcal/mol and in the excited
state is 12.9 and 10.01 kcal/mol for isolated molecule and in ethanol,
respectively. The barriers for interconversion in the excited state
are much higher than that in the ground state (Table 3) and so the
interconversion of isomers I and II is not possible in excited state.
Fig. 6. Fluorescence quenching spectra of BSA at different concentrations of imida-
zole (6).
ent binding constant (KA) were measured. The absorption spectra
of BSA in the presence and absence of imidazole were different
(Fig. 5). The absorption band of 210 nm of BSA is the characteris-
tic of ␣-helix structure of BSA. The intensity of absorbance of BSA
was decreased with increasing concentration of imidazole 6 and the
peak was red shifted. The results indicated that there exists interac-
tion between imidazole (6) and BSA results ground state complex
was formed [31–33].
3.3. Binding interaction of BSA with bioactive imidazole
derivative
Fluorescence quenching spectra (Fig. 6) of solutions containing
BSA fixed concentration and different concentrations of imidazole.
It can be observed that the fluorescence intensity of BSA decreases
regularly with the increase addition of imidazole (6) but there
is no significant emission wavelength shift, suggest that imida-
zole (6) interact with BSA and quench its intrinsic fluorescence. A
Forster type fluorescence resonance energy transfer (FRET) mech-
anism (Fig. 7) is involved in the quenching of Trp fluorescence by
The interaction between bioactive imidazole derivative and
bovine serum albumin (BSA) was investigated using fluorescence
and UV–vis spectral studies and the fluorescence quenching of
BSA by imidazole derivative was the result of the formation of
BSA–imidazole complex. The binding site number (n) and appar-
Fig. 4. (a) PES for the interconversion of the rotamers of 4 in the ground state. (b) PES for the interconversion of the rotamers of 4 in the excited state.

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J. Jayabharathi et al. / Spectrochimica Acta Part A 79 (2011) 1240–1246
Scheme 2. Schematic representation of binding interaction of BSA with PPP.
imidazole 6 in BSA–imidazole (6) complex. This means that the pos-
sible quenching mechanism of fluorescence of BSA by imidazole is
not initiated by dynamic collision but from the formation of the
BSA–imidazole (6) complex (Scheme 2).
3.4. Fluorescence quenching mechanism
The quenching mechanism of imidazole (6) with BSA was
probed using the Stern–Volmer equation [34]. Eq. (1) was applied
to determine KSV by linear regression from the Stern–Volmer plot
of F0/F against [imidazole] (Fig. 9). Dynamic and static quench-
ing can be distinguished by the quenching constant Kq. According
to the literature [35] for dynamic quenching, the maximum scat-
ter collision quenching constant of various quenchers with the
biopolymer is 2.0 × 1010 L mol−1 s−1 and the fluorescence lifetime
of the biopolymers is 10−8 s [36]. From Fig. 8, the values of KSV
and Kq (=KSV/ꢁ0) was calculated. The values of Kq are larger
Fig. 8. Stern–Volmer plot of F0/F against [imidazole].
Fig. 9. Overlapping of fluorescence spectra of BSA with absorption spectra of PPP.
Fig. 7. Fluorescence resonance energy transfer (FRET) mechanism.

J. Jayabharathi et al. / Spectrochimica Acta Part A 79 (2011) 1240–1246
1245
Fig. 10. Synchronous fluorescence spectra of BSA in the presence and absence of imidazole (a) at ꢄꢂ = 15 nm and (b) at ꢄꢂ = 60 nm.
than 2.0 × 1010 L mol−1 s−1, which suggest that the fluorescence
quenching is caused by a specific interaction between BSA and
imidazole (6) and the quenching mechanism mainly arise from
BSA–imidazole (6) complex formation (Fig. S2), while dynamic col-
lision could be negligible in the concentration range studied [37].
The plot of log[(F0 − F)/F] versus log[imidazole] binding constant
KA as 2.52 × 104 M−1 (301 K) and binding sites “n” (1.14) (301 K) of
imidazole with BSA from the intercept and slope.
spectrum may represent that the conformation of BSA is somewhat
changed, leading to the polarity around Tyr residues strengthened
and the hydrophobicity weakened [42]. This is because tyrosine
contains one aromatic hydroxyl group unlike tryptophan and tyro-
sine can undergo an excited state ionization, resulting in the loss of
the proton on the aromatic hydroxyl group. The hydroxyl group can
dissociate during the lifetime of its excited state, leading to quench-
ing. Hence the aromatic hydroxyl group present in the tyrosine
residues is responsible for the interaction of BSA with imidazole.
3.5. Energy transfer from BSA to imidazole derivative
4. Conclusion
The distance between the buried Trp-212 and the interacted
imidazole was estimated by Forster’s non-radiative energy transfer
theory and the overlap fluorescence spectra of imidazole and the
UV–vis absorption spectra of BSA was shown in Fig. 9. According to
Forster’s non-radiative energy transfer theory, the energy transfer
efficiency (E) is defined as the following equations:
Tautomer emission dominates over normal emission in alcohol
at room temperature for hydroxy imidazole. The DFT calculations
indicate in the excited state, the nitrogen atom becomes richer in
electrons than the hydroxylic oxygen atom. This redistribution of
the ␲-electron densities in the excited electronic state is the driv-
ing force for the intramolecular proton transfer from the hydroxylic
group to the nitrogen atom. PES calculation reveals that the barriers
for interconversion in the excited state are much higher than that
in the ground state. So, the interconversions of rotamers are not
possible. The interaction between bioactive imidazole derivative
and bovine serum albumin (BSA) was investigated. The possi-
ble quenching mechanism of fluorescence of BSA by imidazole is
not initiated by dynamic collision but from the formation of the
BSA–imidazole (6) complex.
R06
R06 + r06
F
E = 1 −
=
(3)
(4)
F0
R06 (Å 6) = 8.8 × 1023[k2n−4˚DJ(ꢂ)]
∞
ꢀ
J(ꢂ) =
FD(ꢂ)εA(ꢂ)ꢂ4 dꢂ
(5)
0
FD(ꢂ) is the corrected fluorescence intensity of the donor at
wavelength ꢂ to (ꢂ + ꢄꢂ), with the total intensity normalized to
unity and εA(ꢂ) is the molar extinction coefficient of the accep-
tor at wavelength ꢂ. The Forster distance (R0) has been calculated
assuming random orientation of the donor and acceptor molecules.
In the present case, Ä2 = 2/3, n = 1.334, ˚D = 0.42 and from the avail-
able data, it results that J(ꢂ) = 2.45 × 10−15 cm3 L mol−1, E = 0.34,
R0 = 1.92 nm and r = 2.2 nm were calculated. The donor-to-acceptor
distance is less than the 8 nm, and the long distance indicates that
the quenching mechanism is a dynamic one, which is strong evi-
dence for static quenching and energy could transfer from BSA to
imidazole (6) [38].
Acknowledgments
One of the author Dr. J. Jayabharathi, associate professor of
Chemistry, Annamalai University is thankful to Department of Sci-
ence and Technology [No. SR/S1/IC-07/2007] and University Grants
commission (F. No. 36-21/2008 (SR)) for providing fund to this
research work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2011.04.049.
3.6. Synchronous fluorescence spectroscopic studies of BSA
The synchronous fluorescence spectra [39–41] of BSA with var-
ious amounts of imidazole (6) were recorded at ꢄꢂ = 15 nm and
ꢄꢂ = 60 nm (Fig. 10a and b), respectively. The emission wavelength
of the tyrosine residues is blue-shifted (ꢂmax from 362 to 351 nm in
Fig. 10a) with increasing concentration of imidazole. At the same
time, the tryptophan fluorescence emission is decreased regularly,
but no significant change in wavelength was observed. It suggests
that the interaction of imidazole with BSA does not affect the con-
formation of tryptophan micro-region. The tyrosine fluorescence
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